Microbial Evolution and Systematics

Microbial Evolution and Systematics

• The study of microbial evolution and systematics is crucial in understanding the diversity and relationships among microorganisms
• Topics covered include the earliest life forms, molecular microbial phylogeny, and the identification of bacteria in laboratory cultures and natural environments

The Microbial World and Domains of Life

• All living organisms are grouped into three major domains: Bacteria, Archaea, and Eucarya (algae, fungi, protozoa)
• Organisms within a domain share distinct cellular properties
• Despite their differences, members of different domains share properties and potentially originated from a common ancestor

Molecular Phylogeny Basics

• Phylogeny refers to the evolutionary history of a group of organisms, inferred through nucleotide sequence data
• This inference is based on the idea that bacteria (and all organisms) are related by descent
• The sequence of a bacterium's genome acts as a record of its ancestry
• Evolution involves inherited changes in nucleotide sequences
• Analyzing DNA sequence differences enables the reconstruction of bacteria's phylogenetic history

Genes Used in Phylogenetic Analysis

• 16S rRNA is used in bacteria, and 18S rRNA is used in eukaryotes
• Small subunit ribosomal RNA (SSU rRNA) genes are commonly examined for sequence-based evolutionary analysis because they are universally distributed, functionally constant, sufficiently conserved, and of adequate length
• Carl Woese used SSU rRNA to establish the three domains of life, later receiving the 2003 Craford Prize
• Ribosomal Database Project II holds over 440,000 SSU rRNA sequences
• 23S large subunit rRNA (LSU rRNA) is phylogenetically informative
• Protein synthesis elongation factor Tu (Ef-Tu), heat shock protein Hsp60, and tRNA synthetases are used in phylogenetic analysis

Limitations and Remedies

• SSU rRNA gene sequence variation is limited, which impacts discrimination among bacterial strains
• Studying bacterial evolution using a single gene has drawbacks because the gene may not be present in all microorganisms
• RecA, for example, is present only in bacteria
• Using multiple genes remedies the limitations of using a single gene

Evolutionary Analysis: Methods

• Steps for evolutionary analysis include obtaining DNA sequences, sequence alignment, creating phylogenetic trees, and tree reconstruction

Molecular Phylogenetic Sequence Analysis

• The initial sequence analysis step involves aligning the sequence of interest with sequences from homologous genes from other strains or species

Phylogenetic Trees

• Phylogenetic trees are graphic illustrations showcasing the relationships among sequences
• Trees consist of nodes and branches
• Branches define the order of descent and ancestry
• Branch length represents the amount of changes that have occurred along that branch

Microbial Systematics Fundamentals

• Systematics involves the study of organismal diversity and relationships
• Systematics links phylogeny with taxonomy
• Organisms are characterized, named, and grouped based on natural relationships

Species Concept in Microbiology

• The biological species concept is not applicable because prokaryotes are haploid organisms that do not undergo sexual reproduction
• A prokaryotic species is a group of strains that cluster closely together phylogenetically and are distinct from other groups, based on DNA sequences from multiple genes
• A current definition of prokaryotic species includes strains sharing high similarity in independent traits
• Important features include DNA-DNA hybridization of 70% or greater, and 16S rRNA gene sequence identity of 97% or greater
• 16S rRNA gene sequences act as the "gold standard" for identifying and describing new species
• A bacterium should be regarded as a new species if its 16S rRNA gene sequence differs by over 3% from any named strain
• A bacterium should be regarded as a new genus if its 16S rRNA gene sequence differs by over 5%

Polyphasic Approach

• Traditionally, taxonomy was based on phenotypic characteristics but now includes DNA sequences
• The polyphasic approach now includes phylogenetic, genotypic, and phenotypic analysis: morphological, metabolic, physiological, and chemical characteristics or cell

Phenotypic Analysis

• Organisms are grouped based on similarities and complemented by phylogenetic analysis
• An organism's habitat and ecology are also important characterization criteria

Phenotypic Characteristics with Taxonomic Value

• Morphology is a key factor that includes cell and colony morphology, cell size and shape, gram reaction and flagellation patterns
• PHB granules, gas vesicles, magnetosomes, stalks or appendages Fruiting-body formation are also key morphological characteristics
• Motility analysis includes observation of nonmotile cells
• Gliding, flagellar and or gas vesicle motility are key
• Metabolism assessment includes energy conservation (phototroph, chemoorganotroph, chemolithotroph) and the utilization of individual carbon, nitrogen, or sulfur compounds
• Metabolism assessment also includes fermentation of sugars, growth factor requirements and nitrogen fixation
• Tolerance to temperature, pH, and salt ranges; response to O2 (aerobic, facultative, anaerobic) O2 are aspects fo assessment
• Production of catalase, oxidase and extracellular enzymes are assessed to determine physiology
• Cell chemistry traits include analysis of polar lipids, fatty acids, or respiratory quinones
• Analysis is also given to other trails such as pigments, luminescence serotype and antibiotic sensitivity

FAME: A Key Phenotypic Analysis Tool

• Fatty Acid Methyl Ester (FAME) analysis identifies the types and proportions of fatty acids in the cytoplasmic and outer membranes
• It serves as a major phenotypic parameter.
• FAME is applied in clinical, public health, food, and water evaluations and is commonly used when defining new species

Analysis Workflow

• An organism is isolated from bacterial culture, then fatty acids are extracted, followed by a step to created methyl esters
• Gas chromatography is performed, with resulting peaks compared to patterns in a database to identify the organism

FAME Analysis Drawbacks

• Standardization is vital because fatty acid profiles can vary depending on medium, growth phase and temperature
• FAME analysis is limited to organisms that can be grown matching conditions in the database

Genotypic Analysis Importance

• Provides insight at the DNA level

Genotypic Analysis: DNA-DNA Hybridization

• If two organisms have a high number of similar genes, their DNA should hybridize
• This should exist in approximate porpotions with similarities in the gene sequence, acting as a index of similarity
• Complements 16S rRNA sequencing

How Hybridization Works

• DNA is prepared for two organisms shear and label in the case of organism 1 and shear for organism 2
• Next heat to denature, creating an environment where hybridization can occur as mixed with mix of DNA from 2 organisms with unlabeled amounts in excess

Genotypic Analysis: DNA Profiling

• This method involved creating DNA banding patterns based on repetitive extragenic palindromic PCR (rep-PCR)
• Presence of highly conserved repetitive DNA elements found randomly around the bacterial genome
• Different banding patterns are formed based on the presence and location of these elements which depend on the organism genomic sequence

Genotypic Analysis: MLST

• Multilocus sequence typing (MLST) involves sequencing several different housekeeping genes from an organism
• Afterwards sequences are compared with housekeeping genes, with different strains present on the same organism

Definition of Prokaryotic Species

• A prokaryotic species is defined operationally as a collection of strains sharing a high degree of similarity in several independent traits
• A high degree of similarity is determined through through DNA-DNA hybridization of 70% and greater, as well as 16S rRNA gene sequence of 97% or greater

Classification

• Relies on the organization of organisms into progressively more inclusive groups on the basis of either phenotypic similarity or evolutionary relationship
• Species are made up of several strains
• Similar species are grouped into a genera, followed by: similar genera grouped into families, orders grouped into class, classes grouped into phylum, phylum placed in a domain
• Nomenclature is actual naming of organisms, using formal formal rules
• Following the binomial system of nomenclature, prokaryotes are given genus names and species epitopes: eg, Bacillus subtilis(slender), Bacillus cereus (waxen), *Bacillus megaterium *(big beast)

Regulation of Systematics

• The assignment of names for species and higher groups of Bacteria and Archaea regulated by the Bacteriological Code, also known as The International Code Of Nomenclature of Bacteria
• There is no ‘official’ classification of Bacteria and Archaea, it's based on scientific judgement
• The classification system the ‘Taxonomic Outline of the Prokaryotes’ in the *Bergey's Manual of systematic Bacteriology *is widely accepted

Formal Species Recognition Requirements

• Recognition of a new prokaryotic species requires two main actions
• Action 1. Deposition of a sample of the organism in 2 culture collections
• Action 2. Official publication of the new species name and description in the International Journal of Systematic and Evolutionary Microbiology (IJSEM)

The Role of Culture Collections

• Microbial culture collections store microorganisms as viable cultures as viable cultures ( Frozen or Freeze dried)
• They also are repositories type strains
• Specifically when a new species is described in a scientific journal,a strain is designated as the nomenclatural type of the taxon for future taxonomic comparisons with other strains of that species